This proposal addresses one of the most urgent needs in the clinical management of head and neck squamous cell carcinoma (HNSCC), the development of targeted and less toxic therapies. Recent studies have shown that 50-60% of HNSCC tumors harbor mutations in the TP53 tumor suppressor gene with other studies demonstrating that disruptive mutations in TP53 are associated with worse prognosis and survival. Additional studies have linked HPV infection as a second pathway to p53 inactivation in HNSCC. Despite overwhelming evidence implicating p53 functional derangement in the biology and clinical outcome of HNSCC, there are no targeted therapies that capitalize on this knowledge. Towards this goal, we hypothesized that the driver oncogenic/tumor suppressor mutations (i.e., TP53 loss) that confer dominant malignant phenotypes in cancer cells also engender unique, exploitable vulnerabilities. Since p53 mutant HNSCCs are aggressive tumors incapable of G1 arrest and with higher levels of genomic instability, these tumors rely on a functional G2/M cell cycle checkpoint to repair the DNA damage that might occur as a result of this instability or through genotoxic therapy. In support of this hypothesis, we identified p53 synthetic lethal interactions with several G2/M checkpoint regulators using high throughput arrayed siRNA gene silencing against human kinases in p53-mutated HNSCC. Moreover, treatment with AZD1775, a specific WEE1 inhibitor, blocked tumor growth as a single agent and caused tumor regression when used in combination with cisplatin in p53 mutant HNSCC xenografts. To translate these findings to the clinic, we opened a phase I clinical trial with AZD1775 in combination with neoadjuvant weekly cisplatin and docetaxel in previously untreated, metastatic HNSCC patients. Building on these successes, this proposal will provide needed mechanistic understanding on the biology of p53 alterations and G2/M reliance in HNSCC, while providing translational data to advance WEE1 inhibition with AZD1775 as a novel therapy for HNSCC. Therefore, in Aim 1 we will determine the mechanism(s) of growth arrest upon WEE1 inhibition in HNSCC and determine how p53 inactivation affects this response. In Aim 2 we will identify novel sensitizers to the WEE1 inhibitor AZD1775 to unveil novel synergistic and less toxic partners for combinatorial therapy. Lastly, in Aim 3, we will leverage our ongoing phase I clinical trial to functionally evaluate the effects of AZD1775 on tumor biopsies and relevant preclinical models established from patient biopsies before and after treatment to correlate markers of WEE1 inhibition to p53 status and clinical efficacy for the first time in HNSCC. This new knowledge will help inform which HNSCC might benefit most from this line of therapy and help advance AZD1775 into phase II clinical trials.